The human immunodeficiency virus (HIV) is the infectious agent responsible for AIDS which is currently one of the worlds foremost health problems. The HIV vpr gene, which is one of the viral regulatory genes, has been implicated in the ability of the virus to infect macrophages, a process which both causes and is enhanced by cell differentiation. Macrophage infection by HIV may have and important role in the chronicity of HIV infection and may directly contribute to some of the clinical features of AIDS, such as encephalopathy and dementia. Studies from our laboratory have shown that the vpr gene can affect the proliferation and differentiation of a number of different eukaryotic cell lines. Therefore one of the aims of this proposal is to evaluate the effect of vpr on the differentiation of monocyte macrophage cell lines in vitro. This will test the hypothesis that vpr enhances the ability of HIV to productively infect macrophages by modulating the differentiation of this cell type. The basic approach to these studies will be to transfect either U937 or HL60 cell lines with inducible expression vectors containing the vpr gene or to infect cells with retroviral vectors packaged as amphotropic retroviruses. The resulting cells will then be characterized in terms of the expression of monocyte macrophage differentiation antigens and macrophage function. The use of inducible vectors will allow the isolation of cell lines capable of expressing vpr which will be used for further studies to identify the mechanism of action of vpr. The application of subtractive hybridization techniques to RNA derived from these cells will generate probes for genes that are transcriptionally regulated by vpr. Such probes will be screened for modulation of expression in HL-60 or U937 cells which have been either transfected with vpr, infected with HIV or differentiated with phorbol esters. Tissue specific expression of these genes will be evaluated and from these studies probes will be identified for cDNA's that are likely to be involved in the process of differentiation or the mechanism of action of vpr or HIV. Such probes will then be used to screen cDNA libraries for full length cDNA sequences. Isolated clones will be sequenced and evaluated for use in further studies on the role of vpr in HIV-infection or macrophages. In addition to a role in the virus life cycle it is possible that some HIV encoded proteins may have unwanted secondary effects on the host cell leading to disruption of cellular function. In order to investigate this possibility we have generated transgenic mice containing the vpr gene. Preliminary results suggest that vpr causes premature neonatal death. The second aim of this proposal is to fully characterize this transgenic model of vpr action in vivo. This will involve detailed necropsy and histological analysis and assessment of the degree of transgene expression in different tissues. In addition new transgenic mice will be developed in which vpr expression is restricted to cells expressing CD4. The T lymphocyte function of such animals will then be evaluated. These in vitro and in vivo transgenic models will lead to a clearer understanding of the role of vpr in the life cycle of HIV, particularly the ability to maintain productive infection of macrophages and will also establish if the expression of vpr in vivo contributes to the pathogenesis of HIV induced disease.